Biomatrix Composition and Methods of Biomatrix Seeding

ABSTRACT

Apparatus and methods are described for generating autologous tissue grafts, the apparatus including a point of care SVF isolation unit that includes a tissue digestion chamber in fluid communication with a lipid separating chamber, whereby SVF cells are isolated without centrifugation; and a cell seeding chamber in fluid communication with the SVF isolation unit, said cell seeding chamber adapted to support a cell scaffold. Methods and materials for cell seeding, including through the provision of micro rough scaffold surfaces, are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of, and claims priority to, U.S.application Ser. No. 12/619,977, filed Nov. 17, 2009 and issued Oct. 21,2014 as U.S. Pat. No. 8,865,199, which in turn claims priority to U.S.Provisional Application Ser. No. 61/115,457 filed Nov. 17, 2008, whichis incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates generally to apparatus, compositions and methodsfor the generation of implantable matrices seeded with reparative cellpopulations.

BACKGROUND OF THE INVENTION

The present invention relates generally to tissue scaffolds seeded withreparative cell populations for tissue repair, including myocardialrepair. Without limiting the scope of the invention, its background isdescribed in connection with existing methods and compositions ofimplantable materials for treating physical defects and wound healing.

Roughly 1% of humans are born with an atrial septal defect (ASD) whichpermits a shunt between the right and left atrium. Other deficienciesinclude ventricular septum defects and patent foramen ovale (PFO). Eachof these defects are amenable to treatment by occlusion either by directsurgical techniques in suturing a patch or by placement of an occludernon-invasively. Current occluders and patch materials includenon-absorbable but biocompatible materials such aspolytetrafluroethylene (PTFE or Teflon® patches such as the GORE HELEX®Septal Occluder), woven polyester (Dacron® fabric disk devices such asCardioSEAL® and STARFlex®), stainless steel and polyurethane (i.e. theSideris buttoned devices), nickel titanium shape memory alloys (i.e. theAmplatzer septal occluder constructed of a mesh of Nitinol wires) orcobalt-chromium-nickel alloys (Elgiloy). Although the synthetic patchesare not absorbed, they act as a scaffold onto which normal tissue cangrow and cover the defect, which is essentially “scarred” into placeafter about 3-6 months depending on the conditions of the defect.

For wound healing and reconstructive surgery, existing materials includethe use of synthetic materials as well as biomaterials generated fromhuman and other animal tissues, such as for example the acellular dermalmatrices (ADM) derived from normal human skin (i.e. Alloderm® ADMs, alsodetailed in U.S. Pat. No. 7,358,284). Synthesized biodegradablescaffolds for tissue repair have been introduced for potentialapplications including tissue formation, expansion of host bone cells,cell transplantation, and bioactive molecule delivery. Preformedbiodegradable scaffolds composed of polyglycolic acid (PGA) and polyL-lactic acid (PLLA) have been FDA-approved (i.e. Vicryl® polyglactinwoven mesh). The biodegradable graft material, Dermagraft®, which hasbeen approved for treatment of diabetic foot ulcers, is manufactured byseeding a polyglactin mesh with human fibroblasts which proliferate andcoat the mesh with dermal collagen, matrix proteins, and growth factorsbefore the mesh is cryopreserved.

The field of regenerative medicine has been extensively studying thepotential of cell therapy for repair of injured or diseased tissue. Todate, cells from multiple sources including embryonic stem cells, bonemarrow derived mesenchymal stem cells, peripheral blood derivedendothelial progenitor cells and mesenchymal stem cells, and selectedadipose derived cells have been demonstrated to enhance tissue repair inone or more experimental models. Translation of these preclinicalfindings into a practical therapy is the subject of significantresearch. Since these research efforts are largely based on the premisethat a single cell type, for example a pluripotent or totipotent stemcell, is the best choice for cell therapy, significant effort has beenfocused on identifying and then obtaining the target cell type.

It has been suggested that the post-graft mechanical behavior of ADMcould be enhanced by cell seeding prior to implantation. (Erdag G,Sheridan R L. “Fibroblasts improve performance of cultured compositeskin substitutes on athymic mice.” Burns 30(4) (2004) 322e8; Fuchs J R,et al. “Diaphragmatic reconstruction with autologous tendon engineeredfrom mesenchymal amniocytes” J Pediatr Surg 39(6) (2004) 834-8). Tissueengineering involving the delivery of autologous stem cells andprogenitor cells seeded on scaffolds is currently at the animaldiscovery stage and involves the seeding of scaffolds followed by invitro culture to produce relatively large pieces of tissue prior toimplantation. Such pre-seeded and cultured scaffolds have been shown tobe of value for tissue repair in animal models.

Recent research in the inventor's laboratories has proven that a mixtureof early mesenchymal, multi-potent, lineage committed and lineageuncommitted stem/progenitor cells and fully differentiated cells can beobtained from many body tissue areas. The early mesenchymal uncommittedcells originate from the microvessels within the tissues. For practicalreasons, adipose tissue is a source that is available in most animal andhuman species without disrupting the physiological functions of thebody. It has been reported that adipose derived stromal cells seededonto carrier bioprosthetics facilitated formation of new bone in ananimal model. (Cowan C M, et al. “Adipose-derived adult stromal cellsheal critical-size mouse calvarial defects” Nat Biotechnol 22(5) (2004)560e7).

Typically, cells for matrix or scaffold seeding are isolated from donortissue and cultured for an extended period of time. For example, the FDAapproved Apligraf® skin grafts available from Organogenesis (Canton,Mass.) are manufactured by first forming a bovine collagen matrix whichis plated with cultured human dermal fibroblasts isolated from humandonor skin. Certain aspects of the manufacturing process are disclosedin Bell, U.S. Pat. No. 5,800,537. The donor fibroblasts are cultured onthe collagen matrix for 6 days to form a dermal matrix. Next the dermalmatrix is plated with cultured human keratinocytes to promotedevelopment of a stratum corneum layer. The entire process takes from 20to 27 days prior to packaging. While useful, such a process does notutilize pluripotent cells and is clearly not amendable to a point ofcare process employing the patient's own (autologous) cells.Additionally, recent findings suggest that the cells do not survive longterm and engraft in the recipient patient thus limiting the utility ofthis allogenic cell product (Griffiths M, et al, “Survival of Apligrafin acute human wounds” Tissue Eng 10(7-8) (2004) 1180).

Alternatively, in research applications, bone marrow aspirate cells havebeen obtained from patients and the cells have been held in place orphysically “trapped” on the matrix by an artificial means such as by athrombin induced clot for holding bone marrow aspirate onto anosteogenic matrix. While these methods may have some utility, theyrequire a prolonged treatment program including several surgicalinterventions.

Methods and compositions for the generation of point-of-care cell seededmatrices have not been heretofore available and there continues to be anunmet need for implantable cell seeded matrices that maybe generated ina single procedure. Also needed are methods and apparatus that permitthe isolation of reparative cell populations that are suitable fordirect seeding on to biocompatible matrices.

The present invention provides methods and materials for the focalapplication of reparative cell populations, for example for repair ofdamaged neurons, muscle, tendons, joints and bone structures, repair ofparenchymal organs such as liver, kidney, heart, or brain, and forrepair of skin tissues including in the treatment of burns, hernias, andnon-healing wounds. Methods and materials are provided to retaindesirable cell populations on biocompatible scaffolds and to mosteffectively use the scaffold in conjunction with a fresh cellularpreparation, which avoids a need to culture the cells.

BRIEF SUMMARY OF THE INVENTION

The invention described provides novel methods and apparatus forpoint-of-care isolation of reparative cell populations that does notrely on a cell property of being strongly adherent, as well asbiocompatible matrices that are suitable for loading with the reparativecell populations and implanted without the need for prolonged culturingof the cells or without the need for preselecting cells by plasticadherence.

In one embodiment of the invention, a graft is provided that includes abiomaterial that is resorbable and is seeded with reparative cellpopulation that allows a natural healing including by differentiation ofcells from the population into different lineages depending onpretreatment of the cells and/or placement of the seeded biomaterial inspecialized tissues that influence the differentiation pathway. For onenon-limiting example, when implanted into the heart, certain of thepluripotent stem cells in the population may turn into fibroblasts whileothers may differentiate into specialized cells such as cardiomyocytesand endothelial cells, thus enabling an accelerated healing andremodeling process that most closely resembles a natural process.

In one embodiment, a method of generating tissue grafts is providedincluding the steps of: isolating stromal vascular fraction (SVF) cellsfrom adipose tissue of a mammal, said SVF cells isolated byenzymatically digesting adipose tissue and separating out lipidcontaining cells by floatation, followed by collecting the SVF cellswithout centrifugation; applying the SVF cells to a first scaffold;incubating the SVF cells with the scaffold for less than 2 hours; andremoving unbound SVF cells from the scaffold, thereby generating a cellseeded tissue graft. In one such embodiment, the cell seeded tissuegraft is generated at a point-of-care and is implanted into the mammalwithout culturing the tissue graft whereas in alternative embodiments,the cell seeded tissue graft is cultured to expand populations of cellsseeded on the graft prior to implanting into the mammal. In oneembodiment of the aforementioned seeding step, the SVF cells are pushedinto contact with the scaffold by pressure or by a partial vacuum. Themethods and apparatus of the present invention are particularly usefulin providing autologous tissue grafts.

If desired, a series of seeding steps may be employed wherein theunbound cells from a first seeding step are applied to second scaffold,wherein the second scaffold is adapted for binding of a differentpopulation of cells than the first scaffold, thereby generating at leasttwo tissue grafts, each seeded with a different subpopulation of cells.By different subpopulations it is meant populations that exhibiteddifferent affinity for the two substrates at the time they were appliedalthough it is understood that the different subpopulations may bothcontain at least some cells having similar or identical phenotypicmarkers.

In certain embodiments, the SVF cells are incubated with inductive mediabefore, during or after being applied to the scaffold. For example, theinductive media may be adapted for generation of one or more ofadipocytes, chondrocytes, endothelial cells, hepatocytes, myoblasts,neurons, and osteoblasts.

Preferably, the scaffolds to be seeded are comprised of a biocompatibleor a biodegradable material. Suitable biocompatible materials includebut are not limited to polytetrafluoroethylene, woven polyester, spunsilicone, open foam silicone encased in microporous expanded PTFE,stainless steel, polypropylene, polyurethane, polycarbonate, nickeltitanium shape memory alloys and cobalt-chromium-nickel alloys, andcombinations thereof. Suitable biodegradable materials include but arenot limited to silk fibroin-chitosan, acellular dermal matrices,collagen, polyglactin, and hyaluronic acid.

In certain embodiments, a cell attachment surface of the scaffoldmaterial is characterized by surface irregularities at a periodicity of1-100 μm. In other embodiments, the surface feature micro surfaceirregularities at a periodicity of 5-20 μm. The surface irregularitiesmay be created by treatment of at least one cell attachment surface ofthe scaffold by one or more of mechanical processes including byembossing, blasting, plasma etching, by controlling polymerization ordrying processes, by heat application, by chemical etching, and bycoating or printing. In one embodiment, at least one surface of thescaffold is characterized by a spongy texture formed by subjecting thenascent scaffold material to a partial vacuum during polymerization ordrying.

The cell seeded tissue grafts disclosed herein may be utilized to treatone or more of: wound healing, burns, bone fractures, cosmetic defects,cartilage damage, tendon damage, ulcers, fistulas, hernias, retinaldegeneration, treatment of ischemic disease, nerve injury, aneurysms,bladder wall repair, intestinal injury, and repair and reconstruction ofvessels.

In one embodiment of the invention, one or more adherence agentsselected to promote adherence of desired cell types to the scaffold areintroduced into the seeding chamber before or during cell seeding. Forexample, the adherence agent may be selected from autologous plasma orserum and components thereof, cold insoluble globulin, carboxymethyldextran, iron dextran, and hyaluronic acid and polymers thereof.

Also provided herein are apparatus for generating tissue grafts, saidapparatus including a point of care SVF isolation unit that includes atissue digestion chamber in fluid communication with a lipid separatingchamber, whereby SVF cells are isolated without centrifugation; and acell seeding chamber in fluid communication with the SVF isolation unit,said cell seeding chamber adapted to support a cell scaffold. In oneembodiment, the cell seeding chamber is a dedicated chamber having anupper portion and a lower portion separated by a support member for thescaffold and further comprising at least one inlet port on the upperportion and at least one exit port on the lower portion. The exit portmay in some embodiments be adapted for attachment to a vacuum or a pumpwhereby the SVF cells can be pulled from the upper portion to the lowerportion across the scaffold. In some embodiments, the cell seedingchamber further includes a drain port in the upper portion. In otherembodiments a plurality of seeding chambers are provided, linked inseriatim through a fluid conduit.

In one embodiment of the invention, a tissue graft is provided thatincludes a freshly isolated reparative cell preparation seeded onto abiomaterial, wherein the reparative cell preparation is seeded onto thebiomaterial in an integrated apparatus that is employed to first isolatethe reparative cell preparation and then seed the reparative cellpreparation onto the biomaterial.

In one method of the invention, a method of generating a cell-seeded,biocompatible matrix at the point of care is provided including:isolating a population of cells at the point of care, said cellsincluding multi-potent progenitor cells, endothelial cells, andfibroblasts; conveying the isolated population of cells onto abiocompatible matrix in a seeding chamber; allowing the cells to adhereto the biocompatible matrix at the point of care; and removing cellsthat are unbound to the matrix, thereby generating a cell-seeded,biocompatible matrix suitable for implantation into a patient at thepoint of care. By point of care it is meant at or near to the site ofpatient care, such as for example, in or near the operating suite orbedside. In an example of a point of care procedure, donor tissue isharvested from a patient, desired cell populations isolated, and atissue graft prepared and implanted into the patient, all such stepsoccurring at or near to the site of patient care and at one clinic orhospital visit. In one embodiment of the method, a cell-seeded,biocompatible matrix is provided in less than about 4 hours in anintegrated process at the point of care, wherein the process includesisolation of a heterogeneous reparative cell population and immediatelyseeding the heterogeneous reparative cell population onto thebiocompatible matrix for implantation.

In one embodiment of the invention, a method of producing a cell-seeded,biocompatible matrix at the point of care is provided that includes:providing a biocompatible matrix that is modified to promote celladherence; seeding the biocompatible matrix with a freshly isolatedheterogeneous reparative cell population that contains cells having aplastic-adherent property as well as cells that lack a property ofplastic adherence; forcing the freshly isolated heterogeneous reparativecell population into contact with the biocompatible matrix by appliedpressure, vacuum or electric field. The biocompatible matrix may beoptionally modified to promote cell adherence via chemical or physicalmodification of the matrix, or coating of the matrix with abiodegradable coating. For example, the modification may includetreatment of at least one cell attachment surface of the scaffold by oneor more of embossing, blasting, plasma etching, by controllingpolymerization processes, by heat application, by chemical etching, andby coating or printing. Alternatively, or in addition, the modificationmay include coating with an adherence agent selected from the groupconsisting of: autologous plasma or serum and components thereof, coldinsoluble globulin, carboxymethyl dextran, iron dextran, and hyaluronicacid and polymers thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, includingfeatures and advantages, reference is now made to the detaileddescription of the invention along with the accompanying figures:

FIG. 1 is a flow chart of a reparative cell isolation method accordingto one embodiment of the present invention.

FIGS. 2A and B represent characterization data for reparative cellpopulations isolated according to the process depicted in FIG. 1.

FIG. 3 represents characterization data for freshly isolated reparativecell populations that have not been separated into adherent andnon-adherent populations.

FIGS. 4 and 5 represent characterization data for freshly isolatedreparative cell populations.

FIG. 6 is a figurative diagram of one embodiment of a cell separationapparatus.

FIGS. 7 and 8 are flow charts depicting seeding methods according to twoembodiments of the invention. In FIG. 8 a series of selective seedingchambers are utilized in serial fashion for positive or negativeselection or a combination thereof.

FIGS. 9A and B represent two alternative embodiments of cell seedingchambers.

FIG. 10 depicts removal of a scaffold from a seeding chamber inaccordance with one embodiment of the invention.

FIG. 11 is a Scanning Electron Micrograph (SEM) of an unseeded AcellularDermal Matrix (ADM) at 100× magnification.

FIG. 12 is a SEM of an ADM seeded with adipose derived stromal cells(ADSC) at 100× magnification.

FIG. 13 is a SEM of an unseeded ADM at 1000× magnification.

FIG. 14 is a SEM of an ADM seeded with ADSC at 1000× magnification.

FIG. 15 is a SEM of a silk fibroin-chitosan scaffold (SFCS) seeded withadipose derived stromal cells (ADSC) at 100× magnification.

FIG. 16 is a SEM of a SFCS seeded with ADSC at 1000× magnification.

FIG. 17 is a SEM of a SFCS seeded with freshly isolated SVF cells at1000× magnification.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be employed in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

Increasing evidence suggests that stem cells are residents of amicro-vascular niche, on stand-by for tissue repair as needed. However,with extensive tissue damage, the local pool of stem cells available forrepair is considered insufficient to fully correct the deficiency.Discarded adipose tissue obtained from liposuction procedures contains asignificant number of mesenchymal stem cells accessed via a relativelylow-risk surgical intervention. Adipose tissue is highly vascularizedand is thus a source of endothelial cells, smooth muscle cells, itsprogenitors and of early multipotent mesenchymal stem cells.

Adipose tissue is characterized by the presence of mature adipocytesbound in a connective tissue framework termed the “stroma.” In thepresent invention, “stromal cells” generally refers to cells resident inthe connective tissue of an organ or tissue. Non-limiting examples ofsuch cells include fibroblasts, macrophages, monocytes, pericytes,endothelial cells, inflammatory cells, progenitors and earlyundifferentiated mesenchymal stem cells. Such cells also participate intissue maintenance and repair, typically as supportive cells. The stromaof adipose tissue includes an array of cells that do not include thelipid inclusions that characterize adipocytes. These includepreadipocytes, fibroblasts, vascular smooth muscle cells, endothelialcells, monocyte/macrophages and lymphocytes.

When the connective tissue of adipose tissue is digested, such as withcollagenase, the lipid containing adipocytes can be separated from theother cell types. In 1964, Rodbell reported the use of collagenase todissociate adipose tissue into a cellular suspension that could then befractionated by centrifugation into an upper, lipid-filled adipocytefraction, and a cell pellet comprised of non lipid-filled cells. Thepelleted non-adipocyte fraction of cells isolated from adipose tissue byenzyme digestion has been termed the “stromal vascular cell” or SVFpopulation. (Rodbell M. “Metabolism of isolated fat cells: Effects ofhormones on glucose metabolism and lipolysis” J. Biol. Chem. 239 (1964)375-380).

Heretofore, adipocytes have been separated from the SVF bycentrifugation wherein the adipocytes float and the cells of the SVFpellet. Typically however, the SVF is subject to further processing andselection, including plastic adherence. Cells from the stromal vascularfraction that have been subject to plastic adherence are typicallyreferred to as cultured stromal vascular cells or “adiposetissue-derived stromal cells” (ADSC). Not withstanding other definitionsthat may exist in the art, as used herein, the term “stromal vascularfraction cells” refers to all of the constituent cells of adipose tissueafter enzyme digestion and removal of adipocytes and are not limited toplastic adherent cells.

Researchers have studied the makeup of the stromal vascular fraction ofadipose tissue across a range of disciplines. Typically, the stromalvascular fraction cells that are adherent have comprised the populationthat has been studied in culture. In addition to fibroblasts, thestromal vascular fraction of adipose tissue has been shown to contain,among other cell types, microvessel endothelial cells, vascularprogenitor cells, adipocyte progenitor cells (preadipocytes), andmultipotent progenitor cells. Subsequent to Rodbell's originalisolation, others, using in vitro and in vivo models, identified cellswithin the SVF that could differentiate into adipocytes. These cellswere termed preadipocytes and were identified as plastic adherent cellswithin the SVF. (Hollenberg C H and Vost A. “Regulation of DNA synthesisin fat cells and stromal elements from rat adipose tissue” J. Clin.Invest. 47 (1968) 2485-2498; Van R L R, Bayliss C E, and Roncari D A K“Cytological and enzymological characterization of adult human adipocyteprecursors in culture” J. Clin. Invest. 58 (1976) 699-704.

Using the basic methodology of Rodbell, but capturing endothelial cellclusters on a 30 μm filter versus collection of the entire stromalvascular pellet, it was demonstrated beginning in the 1970's thatmicrovascular endothelial cells could be prepared from human adiposetissue. (Wagner R C and Matthews M A. “The isolation and culture ofcapillary endothelium from epidymal fat” Microvasc. Res. 10 (1975)286-297). Interestingly, the so described “microvascular endothelialcells” from adipose tissue, unlike microvascular endothelial cells fromother tissues, were observed to be adherent to plastic and as such couldbe easily cultured. See e.g. Kern P A, Knedler A, and Eckel R H.“Isolation and culture of microvascular endothelium from adipose tissue”J. Clin. Invest. 71 (1983) 1822-1829; Hewett P W, et al “Isolation andcharacterization of microvessel endothelial cells from human mammaryadipose tissue” In Vitro Cell. Dev. Biol. 29 (1992) 325-331.

Caplan and Haynesworth (Osiris U.S. Pat. No. 5,486,359) describedisolation of pluripotent mesenchymal stem cells from bone marrow usingPercoll gradient separation and plating of the lowest density fractionon plastic. The isolated mesenchymal stem cells were plastic adherentand had fibroblast-like morphology. A panel of monoclonal antibodies wasdeveloped to these cells and including antibodies termed SH2, SH3 andSH4. These antibodies now have the following correlated CD markers: SH2(CD105), SH3 and SH4 (CD73). Davis-Sproul et al. (Osiris U.S. Pat. No.6,387,367) described isolation of pluripotent mesenchymal stem cellsfrom bone marrow or blood using density gradient separation andcollection of the light density cells followed by immunomagnetic beadseparation of CD45+ cells. These cells were also positive for SH3(a.k.a. CD 73) or SH2 (a.k.a. CD 105) and could be pre-selected forthese markers.

Yuan-di Halvorsen (Artecel U.S. Pat. No. 6,391,297) used the SVCisolation technique of Rodbell, to wit, collagenase digestion andcentrifugation, followed by plastic adherence to isolate stromal cellsfrom adipose tissue. The stromal cells were cultured and induced todifferentiate into either adipocytes by the bone marrow stem celldifferentiation method of Hauner, which involved culture in a serum freemedium supplemented with triiodothyronine, insulin and glucocorticoid(J. Clin. Invest. 84 (1989) 1663), or into osteoblasts using osteoplastdifferentiation medium which critically included β-glycerophosphate andascorbate-2-phosphate. Later, Yuan-di Halvorsen described that stromalcells isolated from adipose tissue by collagenase digestion andcentrifugation followed by plastic adherence could be induced todifferentiate into preadipocytes by culture in a medium that criticallyincluded thiazolidinedione followed by culture in a medium criticallyincluding glucose, insulin and glucocorticoid. (Zen-Bio, U.S. Pat. No.6,153,432, filed Jan. 29, 1999). Yuan-di Halvorsen et al (Artecel U.S.Pat. No. 6,429,013) later used the stromal vascular cell isolationtechnique of the above referenced U.S. Pat. No. 6,153,432, to isolateadipose-derived stromal cells that were induced to differentiate intochondrocytes by culture with a differentiation medium that included aglucocorticoid such as dexamethasone and a member of the TGF-βsuperfamily.

The ability of plastic adherent SVF cells to differentiate into multiplelineages fit the criteria of multipotent mesenchymal stem cells. (Seereview by Zuk et al “Human Adipose Tissue is a Source of MultipotentStem Cells” Mol. Biol. Cell 13 (2002) 4279-95). In 2005, theInternational Society for Cellular Therapy (ISCT) stated that thecurrently recommended term for plastic-adherent cells isolated from bonemarrow and other tissues is multipotent mesenchymal stromal cells (MSC)in lieu of the prior “stem cell” term.

As used herein the term Mesenchymal Stromal Cell (MSC) means thedefinition adopted by the International Society for Cellular Therapy andpublished in a position paper by Dominici et al, Cytotherapy 8 (2006)315. In accordance with the position paper, MSC must exhibit:

-   -   1) adherence to plastic in standard culture conditions using        tissue culture flasks;    -   2) a specific surface antigen (Ag) phenotype as follows:        -   positive (≧95%+) for CD105 (endoglin, formerly identified by            MoAb SH2), CD73 (ecto 5′nucleotidase, formerly identified by            binding of MoAbs SH3 and SH4), CD90 (Thy-1), and        -   negative 2%+) for CD14 or CH11b (monocyte and macrophage            marker), CD34 (primitive hematopoietic progenitor and            endothelial cell marker), CD45 (pan-leukocyte marker), CD79α            or CD19 (B cells), and HLA-DR (unless stimulated with            IFN-γ); and    -   3) tri-lineage mesenchymal differentiation capacity: able to        differentiate in vitro into osteoblasts, adipocytes and        chondrocytes in inductive media.

MSC have been traditionally defined as spindle-shaped or fibroblast-likeplastic adherent cells. Although originally isolated from bone marrow,MSC have now been isolated from a variety of tissues including boneperiosteum, trabecular bone, adipose tissue, synovium, skeletal muscle,dental pulp and cord blood.

Adipose-derived stem cells (ADSCs) have been reported to confer benefitsin vivo including as angiogenic agents and in promoting multi-lineagerestoration of soft tissue defects. See Altman A M, et al. “Dermalmatrix as a carrier for in vivo delivery of human adipose-derived stemcells.” Biomaterials 29(10) (2008) 1431-1442; Kim W S et al. “Woundhealing effect of adipose-derived stem cells: A critical role ofsecretory factors on human dermal fibroblasts.” J Dermatol Sci 48(1)(2007) 15-24; and Kim Y, et al. “Direct comparison of human mesenchymalstem cells derived from adipose tissues and bone marrow in mediatingneovascularization in response to vascular ischemia.” Cell PhysiolBiochem 20(6) (2007) 867-876.

However, it has been shown that the phenotype of plastic adherentadipose derived cells changes with cell culture and is influenced byculture conditions. (Gimble J and Guilak F “Adipose-derived adult stemcells: isolation, characterization, and differentiation potential”Cytotherapy 5(5) (2003) 362-369; Boquest A C, et al “Isolation andtranscription profiling of purified uncultured human stromal stem cells:Alteration of gene expression after in vitro cell culture” Mol. Biol.Cell 16(3) (2005) 1131-1141).

As used herein, “reparative cell population” refers to a mixture ofcells that includes “tissue engrafting cells” that are herein defined toinclude MSC as well as cells such as fibroblasts and endothelial cellsthat are able to proliferate and engraft a target tissue when returnedto the body. The reparative cell population may also include one or more“supportive cell” populations. Supportive cells are herein defined ascells that do not permanently engraft in the target tissue but may aidin the tissue remodeling process that is essential to healing of damagedtissue. These may include, for example, lymphocytes and macrophages. Asused herein the term “reparative cell population” is not limited toplastic adherent cells and may be the same as adipose stromal vascularfraction cells under some circumstances.

Advantageously, such reparative cell preparations can be utilized forcell therapy without prior expansion in cell culture. Prerequisite forsuch a procedure is the requirement to obtain a sufficient number ofcells for therapeutic use without expanding the cells in culture.Subcutaneous tissue may provide as many as 300,000 reparative cells pergram, which have an appropriate cell type composition. In contrast tothe prevailing view that a single cell type is optimal for cell therapy,the present inventors believe that multiple cell types are able to actin a coordinated manner to achieve healing and/or repair. Thus, in oneembodiment a heterogeneous reparative cell population is provided tomediate a tissue healing and repair process that emulates endogenousrepair.

In the present invention, “progenitor cells” generally refer touncommitted mesenchymal stem cells in various mesenchymal tissues, suchas muscle, bone, cartilage and adipose tissue and vascular progenitorcells that can be differentiated into vascular cell types. Such cellsare generally believed to constitute a cellular reserve fraction andfunction as target engrafting cells.

The present invention may be utilized in a process for the isolation ofcell populations without loss of cells that would otherwise be usefulbut lack a property of being strongly adherent when first removed fromthe body. For example, when plated onto plastic, adherence ofnon-fibroblast cells in a fresh cell preparation may require severalhours to more than one day. Culture of the fresh cell preparationchanges two characteristics. First, monolayer culture enhances certaincell populations resulting in a cell preparation that is distinct fromthe fresh isolate. Second, culture in an adherent monolayer selects andconditions the cells for adherence, so that upon passaging and replatingthe resulting cell populations adhere much more rapidly (i.e., <30 min).Isolation of MSC involves plastic adherence by definition and eliminatesnon-adherent and weakly adherent cells in spite of their beneficialproperties.

To have clinical utility as a point of care product, the presentinventors believe that cell seeding onto a matrix or scaffold forimplantation would be preferably accomplished in < about 2 hours andmore preferably in < about 1 hours. In other embodiments, matrices areprovided that are adapted to provide rapid, adherence or incorporationof select constituent subpopulations of reparative cells such that cellselection can be performed at the point-of care.

In one embodiment of the invention a method of modifying the surface ofa biocompatible matrix or scaffold is provided to enable selective,rapid adherence of freshly prepared reparative cells, stem cells, orprogenitor cells. In one embodiment the selective adherence occurs in anincubation time frame of < about 2 hours. If desired, followingincubation, non-adherent, undesirable cells are removed with a wash stepprior to implantation of the seeded matrix. In one embodiment of theinvention, modifications such as coating of matrices and/or chemical orphysical modifications are undertaken such that the matrix has increasedselectivity for freshly isolated reparative cells over a contact time of≦1 hour. If desired, unbound cells may be removed by washing. In oneembodiment of the invention, a method is provided that includesassembling a biocompatible matrix to create a three dimensional topologythat enhances selective, rapid adherence of freshly prepared reparativecells, stem cells, or progenitor cells. Rapid adherence is hereindefined as adherence occurring in a time frame of < about 2 hours.

In one embodiment of the invention, the biocompatible matrix comprisesone of more of: collagen, PLGA, PGA, silk fibroin, chitosan,polypropylene, acellular skin preparations of human or other animalorigin, and hyaluronic acid polymers (i.e. HYAFF®-11 sponges). Thematrices may be used without coating or may have surface modificationsincluding coating with specific cell adhesion compounds such ashyaluronic acid, fibrin, collagen, fibronectin, antibodies, aptamers, orthioaptamers, chemical etching such as with NaOH, coatings such asiridium oxide, and/or manufacturing processes that alter the surfacetopology of existing matrices to increase surface roughness or texturalstructure.

The following examples are included for the sake of completeness ofdisclosure and to illustrate the methods of making the compositions andcomposites of the present invention as well as to present certaincharacteristics of the compositions. In no way are these examplesintended to limit the scope or teaching of this disclosure.

Isolation of Reparative Cells from Adipose Tissue:

In contrast to prior isolation methods, the present invention providesfor isolation of reparative cell populations without the use ofcentrifugation or plastic adherence, and which is suitable for use atthe point of care. In one embodiment of the invention, population ofcells for cell transplantation is prepared by dissociating a sample ofdonor adipose tissue into individual cells and small clusters of cellsuntil the dissociated cells and clusters of cells are reduced indiameter to about 1000 microns or less, phase separating the individualcells and small clusters of cells into an aqueous cellular layer and alipid layer without centrifugation, and collecting cells for celltransplantation from the aqueous cellular layer.

In one embodiment of the invention the phase separation is undertaken byintroducing the dissociated cells, including adipocytes, into a lipidseparating unit in an aqueous medium. The lipid and lipid containingadipocytes float upward, thus forming a top lipid layer in the lipidseparating unit while the non-lipid containing or non-adipocyte cellsfloat downward under the influence of normal gravity and are withdrawnfrom under the top lipid layer. In accordance with this method,non-adipocytes can be separated from lipid containing cells withoutcentrifugation.

In one particular embodiment of the invention, as figuratively depictedin the flow chart of FIG. 1, adipose tissue is introduced into adigestion chamber that includes a digestion fluid and an internaldigestion mesh and the tissues and digested cells are recirculatedacross the digestion mesh until the tissue is separated into a digestionmixture that includes individual cells and small cell clusters, followedby phase separating the digestion mixture through an aqueous mediumdisposed in a lipid separation unit. After the phase separationseparates the constituent cells of the digestion mixture on the basis ofdensity in an aqueous medium, desired cell populations are collectedfrom select regions within the lipid separation unit. Isolation ofdesired cell populations is preferably accomplished in a unitary devicewithout a need for centrifugation. In further embodiments, the digestionmixture is filtered over at least one dispersing filter prior to phaseseparating. In certain embodiments the digestion mixture is finallyconveyed through a dispersing head that is disposed within and forms anentry port to the lipid separating unit. The dispersing head furtherdivides clumps of cells within the digestion mixture as the digestionmixture enters the lipid separation unit. The method is particularlysuitable isolation of cells from adipose-containing tissues of human,equine, canine, feline, simian, caprine, and ovine origin.

Various embodiments of the present invention provide a reparative cellpreparation for cell therapy, wherein the cell preparation comprises aheterogeneous mixture of tissue engrafting cells and supportive cellsthat is derived without prior expansion in cell culture. Once derived,cell preparations of the present invention can optionally undergofurther treatment prior to use for cell therapy. For instance, in oneexample, leukocytes within the cell preparation may be removed. Infurther examples, cell preparations of the present invention are seeded(i.e., applied) onto a biocompatible matrix and are then suitable forimplantation at the point-of-care. Such biocompatible matrices caninclude without limitation scaffolds, grafts, sponges, and other wellknown materials that may be surgically implanted into the subject.

Example 1 Reparative Cell Collection Apparatus

FIG. 6 is a schematic depiction of one embodiment of a unitary apparatusfor isolation of stromal vascular cells, wherein the cells are collectedwithout centrifugation. Apparatus 100 includes a digestion chamber 105and a fat separation chamber (a.k.a. lipid separation unit) 140.Digestion chamber 105 generally refers to a housing that can receive andtreat a biological sample and can have various shapes and structures.The depicted digestion chamber 105 includes at least two compartments,predigestion chamber 102 and post digestion chamber 103, separated bydigestion mesh 101. The digestion chamber may optionally include a vent116 that may include a filter 118 to preserve sterility such as, forexample, an ACRODISC brand syringe filter (Pall Scientific). In thedepicted embodiment, the digestion chamber 105 is cylindrical and thepre and post digestion chambers are formed by placement of an inner meshcylinder 101 disposed within the digestion chamber. The porosity of thedigestion chamber mesh is selected based on various desired propertiesincluding but not limited to a size sufficient for small clusters ofdigested tissue to pass through the mesh without rate limiting cloggingof the mesh. In one embodiment the digestion mesh has a plurality ofholes or pores having an opening size of approximately 2-0.5 mm. In oneembodiment found to be effective, the mesh is a nylon mesh having anaverage pore size of approximately 1 mm.

Adipose tissue in extraction fluid or tumescent is introduced via entryport 110 into predigestion chamber 102. The extraction fluid ortumescent is able to drain through mesh 101 and out drain port 127 andultimately to waste port 115 for discard. Valves 137 and/or clamps (notshown) control the pattern of flow, as well as the action of pump 510.After draining of the extraction fluid and optional washing if desired,a digestion buffer is added to the predigestion chamber via a fill portsuch as fill port 112 and a digestion enzyme or cocktail of enzymes isadded to the predigestion chamber. The enzyme can be added together withthe digestion buffer if desired. In one embodiment found to beeffective, the buffer solution utilized was a lactated Ringer'ssolution, however other physiologic buffers are suitable and are readilyenvisioned by one of skill in the art. In the depicted embodiment, theenzyme may be added through a dedicated port such as fill port 112,which may be constructed in any number of ways including for example asa valvable opening or as a self-sealing septum. Optionally, a compoundsuch as a poloxamer may be added to improve flow and as an aid inmaintaining cell viability. For example, poloxamer 188 may be used atconcentrations ranging from about 0.05% (w/v) to about 5% (w/v).Further, heparin or low molecular weight heparin may be added atconcentration ranging from 1-100 U/ml, preferably between 10-30 U/ml, toreduce formation of clot like clumps and recovery of a unicellularsuspension.

A digestion period is then begun wherein the digestion mixture isrecirculated, typically through the action of a pump such as for exampleroller or peristaltic pump 510. The direction of flow is frompredigestion chamber 102 through digestion mesh 101, into post digestionchamber 103, out drain port 127, and back around into the predigestionchamber through recirculation port 113. This configuration providesample volume for both chambers and, as can be seen by the depictedarrows, the digestion mixture is able to circulate around as well asthrough the digestion mesh 101.

As part of the recirculation loop the digestion mixture may be passedthrough a heat exchanger loop 136 by the action of pump 510. In apreferred embodiment, equipment such as pump 510 and heating element520, shown surrounded by dashed lines, are adapted to be operablyattached to apparatus 100 via tubing but are part of a reusable baseunit that constitutes capital equipment in contrast to apparatus 100,which is designed for clinical use to be a disposable unit that does notrequire any electrically operable components and can be supplied as apresterilized single use unit. The heat exchanger loop 136 is heated byheating element 520 which provides controlled heating to the heatexchanger loop for optimum enzyme activity. As digestion continues anincreasing greater proportion of the adipose tissue is able to cross thedigestion mesh 101. In a preferred embodiment, the apparatus 100 isagitated by shaking during the digestion period. After the adiposetissue is sufficiently digested, the recirculation loop is ceased andthe digestion mixture is directed to fat or lipid separation unit 140.In alternative embodiments, modulation of processing temperatures, forexample to control the activity of digestion enzymes, is provided byenclosing the processing apparatus in a thermally controlled chamber.Such a thermally controlled chamber may be used together with, or inlieu of, use of one or more heat exchanger loops.

In one embodiment, prior to allowing the digestion mixture to enter thelipid separating unit, the unit is prefilled with a separation buffer.In further embodiments of the present invention, various compositionsmay be introduced into a lipid separating unit to aid in phaseseparation. For instance, the separation buffer may comprise separationfacilitating compounds that may be introduced into lipid separating unit140.

The separation buffer may be added through various mechanisms. In thedepicted embodiment, a fill port 112 is provided for separation bufferaddition. In the lipid separation unit 140, phase separation occurs andthe lipid and lipid containing cells float up through the separationbuffer as depicted by the upward directed thick dashed arrow andultimately form a floating lipid phase. The non-lipid containing cells,including a reparative cell population, settle down. After a desiredperiod wherein the lipids have had time to migrate to the top of thechamber, the underlying phase is removed via collection port 172. In theembodiment provided in FIG. 6 flow patterns are depicted with therecirculation of tissue during digestion depicted in solid lined arrowswhile the digested mixture is depicted in dashed lined arrows. In thedepicted embodiment, apparatus 100 further includes a dispersing head168 having a plurality of pores 169 as the entry port of fat or lipidseparating chamber 140. In one embodiment, the average pore size of thedispersing head is in the range of about 0.3 mm (300 microns) to about 1mm (1000 microns), while in another embodiment the average pore size isfrom about 0.4 mm (400 microns) to about 0.6 mm (600 microns). In oneembodiment, the dispersing head has an average pore size of about 0.5 mm(500 microns).

In the depicted embodiment of FIG. 6, dispersing head 168 is asubstantially rigid structure designed to be located relatively close tothe bottom of the lipid separating unit 140. As depicted, the dispersinghead can be directed with its exit openings or pores 169 facing downwardsuch that the fluid flow entering lipid separating unit 140 is in theopposite direction of the buoyancy of lipid-filled cells and thusfurther reduces clumps and releases reparative cells trapped togetherwith lipid-filled adipose cells. Use of the dispersing head has beenshown by the present inventors to result in greater yield of reparativecells.

In the embodiment depicted in FIG. 6, a further dispersing filterchamber 165 including dispersing filter 170 is included in-line prior tothe dispersing head 168 and is adapted to further divide clumps of cellsand to protect the dispersing head from clogging. In one embodiment, thedispersing filter is dimensioned to have a pore size ranging from about0.2 mm (200 microns) to about 0.3 mm (300 microns). In yet anotherembodiment, the dispersing filter has an average pore size of about 0.25mm (250 microns). However, one of ordinary skill in the art willrecognize other suitable filter sizes that can be used in the presentinvention. Furthermore, one of ordinary skill in the art will recognizethat dispersing filter 170 can be in other forms or may, in someembodiments, be eliminated entirely depending on the configuration ofthe apparatus.

Likewise, one of ordinary skill in the art will recognize that container105 can have various other shapes and arrangements. As with otherembodiments, digestion chamber 105 is in fluid communication with thelipid separating unit 140, and any intervening filters, via a tubingnetwork. The pattern of flow is controlled by one or more valves 137and/or clamps 138 as well as the action of the pump. The embodimentdepicted in FIG. 6 includes a separate waste line 115.

As depicted in FIG. 6, the upper most portion 164 of the lipidseparating unit may have a greater diameter than the lower portion toaccommodate the floating fat layer. The embodiment depicted in FIG. 6also includes an optional seeding chamber 180, which may include a cellseeding substrate or scaffold 185. Such a seeding chamber may servevarious functions. In one embodiment, the chamber can contain theaforementioned substrate or scaffold on which cells might be seeded asliquid is drained from the lipid separating unit. In operation,reparative cells collected using the apparatus can be directly disposedonto the cell seeding substrate and either implanted on the patient orremoved to an incubator for further cellular expansion. In anotherembodiment, the chamber might be adapted to allow buffer exchange. Infurther embodiments of the present invention, chamber 180 may beentirely absent or may be provided as a separate apparatus. In some ofsuch embodiments, lipid separating unit 140 may have a port and/or anopening for passage of separated material.

In another embodiment, various chamber and compartment of the apparatusmight contain materials such as antibodies or aptamers or thioaptamersthat could be used to negatively select for materials to be removed fromthe processed material for further purification. Cell selection agentsthat may be introduced into containers of the present inventiongenerally refer to one or more compounds for positive cell selection ornegative cell selection. For negative cell selection, such cellselection agents may aid in the depletion of various cells from abiological sample, such as the depletion of leukocytes and/orerythrocytes in one embodiment. For positive cell selection, the cellselection agents may specifically bind a desired cell type forisolation. Cell selection agents suitable for use in the presentinvention may include, without limitation, an antibody (see U.S. Pat.Nos. 6,491,918, 6,482,926, 6,342,344, 6,306,575, 6,117,985, 5,877,299,and 5,837,539), an aptamer (see U.S. Pat. No. 5,756,291), and/or athioaptamer (see U.S. Pat. No. 6,867,289), for example, all of which areincorporated herein by reference in their entirety. In some embodiments,the cell selection agents may also be immobilized on the matrix orscaffold 185.

As depicted in FIG. 6, the lipid separating unit may optionally includefill port 112 and a vent port 116 with sterility filter 118. Anadditional collected cell filter 175 may be optionally included prior tothe seeding chamber 180 and may be adapted to optionally provide forpurification and sizing of desired cells as well as to prevent cloggingof downstream components. Collected cell filter 175 is generally acircular structure in the present example, though a person of ordinaryskill in the art could envision other shapes and structures.

In the example shown in FIG. 6, collected cell filter 175 is desirably afilter with a pore size of less than about 250 microns. However, inother embodiments, collected cell filter 175 can have a pore sizeranging from about 0.01 mm (10 microns) to about 0.1 mm (100 microns).In another embodiment, collected cell filter 175 can have a pore sizeranging from about 0.03 mm (30 microns) to about 0.05 mm (50 microns).In a further embodiment, the average pore size in collected cell filter175 is about 0.037 mm (37 microns). In additional embodiments, thecollected cell filter may be entirely absent.

Example 2

In one example, a reparative cell population was isolated as follows.Lipoaspirate was collected under informed consent in the operating roomdirectly into a unitary purification apparatus by standard suctionassisted lipoplasty with tumescent. The apparatus including tumescentfill was transported to the laboratory and processed within 2 hours ofcollection. In practice however, it is anticipated that the purificationapparatus will be suitable for, and will be used, in the operatingsuite. The digestion chamber of the apparatus as depicted in Example 1included a predigestion chamber and an inner postdigestion chamberseparated by a nylon mesh having a pore size of approximately 1 mm. Thetumescent was drained and a volume of approximately 100 ml of drainedlipoaspirate was washed by draining the predigestion chamber andrefilling with a solution of lactated Ringer's solution, which wasprewarmed to 37° C. containing a proteolytic enzyme combinationcomprised of collagenase IV (60,000 U) and dispase (120 U). Anadditional 150 ml of lactated Ringer's was added to the lipid separatingunit. The digestion recirculation loop was implemented by a pumpactuated flow path from the predigestion chamber into the postdigestionchamber and including passage across a heat exchanger that maintains thedigestion mixture at approximately 37° C. Recirculation was continuedfor approximately 30 to about 60 minutes or until greater than 90% ofthe cellular volume of the predigestion chamber was able to pass the 1mm mesh into the post digestion chamber. The design of the pre and postdigestion chambers, separated by the nylon mesh across which therecirculation flow path passes repeatedly, provided trapping ofconnective and other debris tissue on the digestion mesh. Afterdigestion was sufficiently complete, the digestion mixture was pumpedtangentially over a nylon dispersing filter having a pore size of 250μm. The filtered digestion mixture was then pumped into a columnar lipidseparating chamber that was integral to the apparatus. As previouslymentioned, the lipid separating chamber was prefilled with a volume of150 ml lactated Ringer's solution prior to introduction of the digestionmixture such that when the filtered digestion mixture entered thechamber, any clusters of cells including lipids or adipocytes, weresubject to fluid shear as the lipid moieties float upward in the aqueoussolution. The filtered digestion mixture entered the lipid separatingchamber through a dispersing head having a plurality of downwardlydirected pores with a pore size of 500 μm and disposed proximally to abottom inner surface of the lipid separating unit. The design wasadapted for forcibly flowing the cell mixture against an inner surfacewithin the lipid separating unit and thereby further disrupting cellclusters within the cell mixture prior to fluid phase separation. Fluidphase separation was allowed to proceed at room temperature for about 5to about 30 minutes prior to collection of the stromal vascular fractionfrom the bottom of the lipid separating chamber.

Example 3

After processing tissue in the device, cell viability and cell numberwere determined. In one processing run, the collected cells were platedat a density of approximately 7×10⁵ cells/cm² into a T185 flask in MEM,20% (v/v) FBS including and antibiotic/antimycotic and culturedovernight at 37° C. in a humidified 95% O₂, 5% CO₂ atmosphere. Afterovernight, non-adherent cells were harvested by aspiration, and adherentcells were harvested by trypsinization. Immediately after harvest, cellswere processed for flow cytometry. Numbers represent the net percentagepositive cells after subtraction of background (2′ Ab only) and gatingto remove debris. FIGS. 2A and B represent data from two processingruns.

Cells collected as described in Example 2 have also been characterizedby direct analysis without separation into adherent and non-adherentpopulations. The results are depicted in FIG. 3.

In comparing the cells isolated as disclosed herein with mesenchymalstromal cells isolated using centrifugation and plastic adherence inaccordance with conventional preparation methods, several notabledifferences are apparent. Mesenchymal stromal cells have beenclassically isolated from adipose tissue using enzymatic digestion,centrifugation to remove lipid filled cells and plastic adherence withculture in vitro. These cells show a fibroblast-like morphology.Although the cells are initially heterogeneous, the phenotype ofpopulation changes in culture including loss of CD31+, CD34+, CD45+cells, and an increase in CD105 and other cell adhesion type molecules.Generally, <10% of the cells express markers associated with stemness(e.g., CXCR4, sca-1, SSEA-4) and a substantial fraction differentiatesinto adipocytes in inductive media. A lesser fraction differentiatesinto other lineages (bone, cartilage, nerve) in inductive media.

The reparative cell population isolated as disclosed herein withoutcentrifugation or plastic adherence is also a heterogenous populationand generally <10% express markers associated with stemness (e.g.,CXCR4, Sca-1, SSEA-1, SSEA-4, VEGFr2, CD117, CD146, Oct4). However, asubstantial fraction of the early multipotent stem cells are not plasticadherent. Importantly, a substantial fraction of cells expressingmarkers of stemness, endothelial cell lineages and/or exhibiting a smalldiameter 6 mm) are not adherent and are lost using conventionalisolation methods that rely on plastic adherence or centrifugation.

Example 4

Cells were collected as essentially described in Example 2. Digestionwith warming, agitation, and recirculation was conducted for 30 minutes.The resulting slurry was then pumped through a 250 μM filter and intothe lipid separating unit. After a 10 minute static hold, the loweraqueous phase was collected, and cells from this phase were concentratedby centrifugation at 400×g before characterization. Cell yield wasdetermined by counting with a hemacytometer. Cell viability was assessedby two assays, trypan blue exclusion using phase contrast microscopy andthe Live/Dead assay (InVitrogen, Inc) using a Coulter Epics XL-MCLcytometer.

Collected cells were characterized by cytometry using cell surfacemarkers CD31, 34, 44, 45, 71, 73, 90, 105, 117, 146, SSEA-4, and Sca-1.All assays were performed using a Coulter Epics XL-MCL cytometer. Cellpreparations were plated in standard growth medium and culturedovernight in a humidified, 37° C., 95% O₂, 5% CO₂ environment.Non-adherent cells were removed by pipette, and adherent cells weredetached by trypsinization. Cells were layered onto ficoll andcentrifuged at 1000×g to remove erythrocytes prior to incubation withprimary antibody. Adherent and non-adherent cell populations wereanalyzed separately, and then an estimate for surface marker profile inthe total population was calculated from cell counts and surface markerprofiles in the adherent and non-adherent populations. All assays wereperformed with murine anti human antibody specific for the target inquestion and FITC conjugated goat anti murine secondary antibody.

Culture characteristic assays were performed as follows. Fordetermination of doubling time, 1×10⁶ adherent cells from overnightculture were seeded in a 75 cm² flask and cultured for 4˜5 days withmedium changed every 2 days. Cells were harvested by trypsinization andcounted with a hemacytometer. Doubling time was calculated based on cellcount versus the number of cells plated. For determination of colonyforming units (CFU), 3.1×10⁵ cells were suspended in 3.1 ml growth media(MEM, 20% FBS, antibiotic/antimycotic). Duplicate dilutions of cellswere prepared and plated at approximately 2.8×10⁴/cm², 0.5×10⁴/cm², and0.1×10⁴/cm² in 6 well plates. Cells were maintained in a humidified 37°C., 95% O₂, 5% CO₂ environment, and media was changed 2×/week. Colonyforming units were scored after 7-14 days in culture. Cells were fixedand stained with hematoxylin and pictures were taken under themicroscope from 5 fields per well at 25× magnification for quantitation.Colonies with at least 10 fibroblast-like fusiform cells clustered orpiled together were counted. For colonies on the edges of a microscopicfield, only those that were judged to be more than 50% within the fieldwere included in the calculations. Percent colony forming units wascalculated from the number of colonies relative to the total number ofcells plated and averaged across the three dilutions.

Results for subject demographics, cell yield, doubling time and CFU arepresented in FIG. 4. Mean total cell yield was 30×10⁶ cells from the 8tissue samples processed in the device described in Example 1. Cellyield for the two samples from male subjects averaged 13.5×10⁶ whereascell yield from the 6 female subjects averaged 36.9×10⁶. Mean cellviability was 82 and 83 percent for the two respective assays. Meanpercent CFU was 12%, ranging from 6.6 to 15.4.

Results for surface markers are presented in FIG. 5. Surface markers forendothelial cells (CD31), hematopoietic progenitor cells (CD34),leukocytes (CD45), mesenchymal stromal cells (CD44, CD73, CD90) andprogenitor cells (CD117, CD146, Sca-1, and SSEA-4) were observed in allspecimens. The high yield and viability of the diverse population isconsidered by the inventors to be important contributions provided bythe method and apparatus of the invention.

Scaffolds

In one embodiment of the invention, reparative cells are localized ontoa scaffold such that upon implantation a locally high concentration ofreparative cells, including stem cells, is retained at the implantationsite. A number of biocompatible materials including biodegradablematerials are known. Available biocompatible materials includepolytetrafluoroethylene (PFTE), woven polyester (i.e. Dacron® fabric),open foam silicone encased in microporous expanded PTFE (Evera Medical),stainless steel, polypropylene, polyurethanes, polycarbonates, nickeltitanium shape memory alloys (i.e. Nitinol) and cobalt-chromium-nickelalloys (Elgiloy). Although the synthetic patches are not absorbed, theyact as a scaffold onto which normal tissue can grow and cover thedefect, which is essentially “scarred” into place after about 3-6 monthsdepending on the conditions of the defect. In addition to non-absorbablebiocompatible materials, there is a whole range of degradable andbio-absorbable biomaterials that are suitable.

In one embodiment, biomaterials are seeded including, for example, silkfibroin-chitosan, acellular dermal matrices (ADM) derived from normalhuman skin, preformed biodegradable scaffolds composed of polyglycolicacid (PGA) and poly L-lactic acid (PLLA) (i.e. Vicryl® polyglactin wovenmesh), and bioabsorbable scaffolds coated with extracellular matrix(i.e. Dermagraft® polyglactin woven mesh coated with extracellularmatrix (ECM) laid down by fibroblasts prior to cryopreservation). In oneembodiment of the invention, a bioabsorbable three-dimensional scaffoldcomposed of ECM is used as the cell scaffold for the freshly isolatedreparative cells of the invention.

Treatment of the surface of the scaffold, such as with the above NaOHetching, can increase the adherence of stem cells. It has beendetermined by the present inventors that a local micro-roughness and thecreation of niches increases not only the adhesion but also enhances thethree dimension incorporation of stem cells into the material. Themicro-roughness of the surface structure can be increased by mechanicalprocesses including by embossing, blasting, such as particle blasting,or by plasma etching, controlling the polymerization processes, heatapplication, by chemical etching, and including by printing such as byinkjet printing.

Where it is desired to increase the surface roughness of the matrix, amicro-rough surface is generated wherein three dimensional mounds ofscaffold material or peaks, valleys and voids are arrayed to providesurface irregularities at a periodicity of 1-100 μm. In one embodimentthe three dimensional mounds are formed as ridges while in otherembodiments the three dimensional mounds are hemispherical. In otherembodiments a pattern of ridges and hemispherical mounds is formed.Alternatively, or in addition, a spongy surface characterized by voidsis provided.

In one embodiment of the invention, scaffolds are provided that arecharacterized by a spongy texture formed by subjecting the nascentscaffold material to a partial vacuum during polymerization or drying.Desolved gases in the uncured scaffold solution expand and form bubblesthat are induced to rupture just as curing is completed thus forming aplurality of voids and pockets that together result in a sponge likecomposition having a vastly increased surface area. In one embodiment,the formation of bubbles is enhanced by inclusion of a frothing agentinto the uncured scaffold material such that bubble formation isenhanced by application of the vacuum.

In the case of printing such as with inkjet type printing, an array canbe deposited according to a programmed pattern including patternsforming a plurality of surface structures. In other embodiments,printing such as with inkjet type printing is further utilized to applya plurality of different substrate materials and/or growth factorsarrayed on the surface thus promoting the growth of disparate cell typesin a virtual tissue pattern.

Collagen:

Collagen is another biomaterial that is suitable for use with mixedreparative cell populations, both for the process for the selection andadhesion of stem cells, and as a local carrier and matrix or as anadhesion matrix when coated onto other materials. FDA approved Type 1collagen products, such as those available from Collagen Matrix, Inc.,Franklin Lakes, N.J., are commercially available. Such collagens areespecially useful for external application such as non-healing woundsand burns, soft tissue defects, cosmetic surgery, and for nerve repairwherein a collagen sleeve serves as a conduit between the interruptednerve ends.

Bioresorbable Silica Gel Matrix:

In another embodiment, a very recently developed bioresorbable silicagel matrix is seeded with fresh reparative cells. The matrix of silicagel fibers, developed by Dr. Jörn Probst and Dipl.-Ing. Walther Glaubittat the Fraunhofer Institute for Silicate Research ISC in Würzburg, isshape-stable, pH-neutral and 100 percent bioresorbable. The fibers areproduced by means of wet-chemical sol-gel process in which atransparent, honey-like gel is produced from tetraethoxysilane (TEOS),ethanol and water in a multi-stage, acidically catalyzed synthesisprocess. The gel is processed in a spinning tower which produces fineendless threads which are collected on a traversing table and spun in aspecific pattern to produce a multi-layer textile web which can be cutto desired size and sterilized prior to loading into a seeding chamber.

Highly pliable biomaterials loaded with reparative cells can be locallywrapped around the tissue or inserted into a tissue defect where it isdesirable to localize cells for repair at an increased localconcentration. This is especially useful where the scaffold can beintegrated surgically into the defect when carrying cells for nerverepair, wrapping around non-healing bone fractures, ruptures or injuriesto tendons, which normally show a very slow rate of healing,applications in repair of cartilage defects in joints or of scars andinjuries to skin and the underlying tissue and also for plastic,cosmetic, aesthetic repair. In addition, a repair such as anentubulation, or a wrapping around of venous structures, lymphaticvessels, and nerves is also a target of the cell loaded scaffoldsdisclosed herein.

Acellular Dermal Matrix Scaffolds:

In one embodiment of the invention, acellular dermal matrix (ADM)pre-seeded with reparative cells prior to implantation is provided fortreatment of soft tissue injuries including abdominal wall compromiseand soft tissue loss secondary to traumatic and oncologic processes.Human acellular dermal matrix (ADM) has become widely used in plasticsurgery because it is non-immunogenic, mechanically robust and hasfavorable handling characteristics. Recent reports have suggested thatthe post-engraftment mechanical behavior could be enhanced by cellseeding. (Erdag G, Sheridan R L. “Fibroblasts improve performance ofcultured composite skin substitutes on athymic mice” Burns 30(4) (2004)322e8; Fuchs J R, et al. “Diaphragmatic reconstruction with autologoustendon engineered from mesenchymal amniocytes” J Pediatr Surg 39(6)(2004) 834e8). Additionally, others have shown that seedingadipose-derived stromal cells on a carrier bioprosthetic materialfacilitated new bone formation in the treatment of a bony defect. (CowanC M, Shi Y Y, Aalami O O, Chou Y F, Mari C, Thomas R, et al.“Adipose-derived adult stromal cells heal critical-size mouse calvarialdefects” Nat Biotechnol 22(5) (2004) 560e7). Thus, a cell-seeded matrixis believed to offer some benefits for introducing cells to the localenvironment as it provides a framework for the support of theirregenerative capacity. ADM provides a three-dimensional scaffold intowhich seeded cells could incorporate and help to build the foundationfor the integration of local tissue with the graft.

Example 5

Human adipose tissue was obtained from elective body contouringprocedures and the tissue digested with a solution of 0.07% Blendzyme 3(F. Hoffman-La Roche Ltd, Basel, Switzerland) with mild agitation at 37°C. for 60 minutes. The digest was passed through a 100 μm filter, thenthrough a 40 μm filter and finally though a 10 μm filter. The filteredmaterial was centrifuged at 1500 RPM for 10 minutes and resuspended in1×PBS. Following a second centrifugation the cells were resuspended inMEM containing 20% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100μg/ml streptomycin and selected based on adherence to T75 tissue cultureflasks for 24 hours after which non-adherent cells and debris werediscarded by aspiration. Adherent cells were incubated in a 5%CO₂-containing chamber at 37° C. with medium changed every 3 days. ADSCsbetween passages 1 and 6 were used for all experiments.

For seeding of the grafts, six-mm diameter ADM pieces (Alloderm®Lifecell) having a thickness of 0.53-0.76 mm were placed completelycovering the well bottom in 96-well plates with the papillary dermalsurface facing up and the grafts were covered with 200 μl aliquots ofmedium alone in the ADM group and with equal volume of cell suspensioncontaining 1×10⁵ ADSC in ADSC-ADM group. Grafts were incubated understandard culture conditions for 24 hours after which overlying medium orcell suspension was aspirated. The grafts were flipped to place theopposite reticular dermal surface facing up, and the correspondingmedium or cell suspension solution was placed on the other side. Graftswere then incubated for 24 hours and transferred to the operating suitefor surgical engraftment.

FIG. 11 is a scanning electron micrograph (SEM) of unseeded ADM at amagnification of 100×. As can be seen in FIG. 11, the ADM ischaracterized by a surface having three dimensional mounds and ridges ata periodicity of 10-100 μm. FIG. 12 is a SEM of the ADM seeded withADSC. As can be seen in FIG. 12, the seeded cells spread out on therough surface. FIG. 13 represents a 1,000× magnification of unseededADM, wherein it can be seen that the ADM not only presents a surfacehaving three dimensional mounds and ridges at a periodicity of 10-100 μmbut also presents surface irregularity on a scale of 1-10 μm. FIG. 14represents the ADM at 1000× magnification now seeded with ADSC where itcan be seen that the ADSC are spread out and adherent to the surface ofthe matrix.

Once on the operative field, grafts were transferred to a sterile 6-wellplate and washed gently in 2×500 μl aliquots of PBS to remove anynon-adherent cells or medium. For the main study groups of seven athymicnude mice were randomized to one of three treatment groups: no graft,ADM alone or ADSC-ADM. Animals in each group received one 6 mm punchlesion and a graft-based repair depending on group randomization.

Human adipose-derived stem cells have been well characterized withregard to profile of expressed surface cluster of differentiation (CD)markers. The ADSCs were negative for the pan-leukocyte marker CD45,separating them from the hematopoietic lineage. They were also negativefor the integrin CD11b (alpha-M chain), an adhesion moleculecharacteristically found on macrophages and leukocytes. The ADSCs werepositive for CD44 (99±1%), CD90 (98±3%), CD105 (98±2%).

Analysis of wound healing rates (rate of wound contraction) was definedas the gross epithelialization of the wound bed. A statisticallysignificant increased rate of epithelialization in the ADSC-ADM groupcompared to the no-graft and ADM groups was noted at postoperative day7. Percent wound closure at post-op day 7 was 56±21% in the no-graftcontrol group, 57±21% in the ADM group and 77±4% in the ADSC-ADM group(p≦0.05). Closure of wounds in the ADSC-ADM group still wassignificantly greater than in the no-graft group at post-op day 10,although the differences between the three groups diminished over timeas would be expected. Because the ADSC were transfected with a vectorexpressing Green Fluorescent protein (GFP) prior to seeding of thegrafts, the status of the ADSC could be monitored during wound healing.It was found that the ADSC actively proliferated post transplantationand could be detected in the graft at 28 days, almost two weeks aftercomplete wound closure. Certain of the ADSCs engrafted into thecutaneous wound milieu via the ADSC-ADM construct demonstrated amicrovascular endothelial phenotype by 2 weeks postoperatively,contributing directly to the establishment of a vascular network in thecontext of tissue regeneration. No GFP expressing cells were detectableat locations 2 cm from the graft, nor in the spleen, liver or kidneys,indicating that the transplanted cells were locally persistent to thesite of engraftment.

The findings demonstrated that a construct created by the seeding ofadipose-derived stem cells upon human dermal matrix could be used as aneffective delivery vehicle in vivo. Furthermore, the use of an ADSC-seeded ADM construct significantly enhanced the rate of wound healingat postoperative day 7. Finally, it was demonstrated that humanadipose-derived mesenchymal stem cells delivered via an ADSC-ADMconstruct persist locally and do not distribute systemically, providinganatomically directed support to tissue regeneration at the desired siteof surgical engraftment.

As an important finding of this study, it was shown that humanadipose-derived stem cells delivered via dermal matrix differentiatedinto derivatives of two germ layers in the setting of this murinecutaneous wound healing model. Ectoderm derivatives were noted at 4weeks at which time GFP positive stem cells were seen to co-localizewith stain against cytokeratin 19, an element of epidermal epithelium,indicating differentiation into epidermal epithelial cells. Spontaneousmesoderm-derivative differentiation patterns were evidenced byGFP-positive ADSCs co-staining with HSP47, an indicator of afibroblastic differentiation fate. Further evidence of mesodermaldifferentiation paths was observed with the identification of smoothmuscle actin (SMA)-positive and von Willebrand Factor-positive engraftedGFP cells, appearing structurally integral to and associated withneo-vascular structures. This finding is consistent with the recentreport by Wu and colleagues on the ability of 1×10⁶ mesenchymal stemcells delivered to the cutaneous wound to differentiate into variousdermal appendages. (Wu Y, et al. “Mesenchymal stem cells enhance woundhealing through differentiation and angiogenesis” Stem Cells 25(10)(2007) 2648-59).

Consistent with the observed enhanced rate of wound healing, acircumferential halo of hyperemia was noted at the margins of thehealing wounds in the ADSC-ADM group, observed most prominently atpostoperative day 7, which was not seen in the no-graft or ADM groups.Kim and colleagues recently reported on markedly enhanced rates of grosswound closure in athymic mice treated locally with a dose of 1×10⁶ADSCs. (Kim W S, et al. “Wound healing effect of adipose-derived stemcells: a critical role of secretory factors on human dermal fibroblasts”J Dermatol Sci 48(1) (2007) 15-24). The present study indicates thatwith the use of a carrier system a therapeutic effect is facilitatedwith only a fifth of the cells (2×10⁵).

Silk-Chitosan Scaffolds:

In one embodiment of the invention, a particularly treated blend of silkfibroin and chitosan is provided that is pre-seeded with reparativecells prior to implantation. The fibers of silkworm silk consist of twomain proteins, fibroin, which is the structural center of the silk fiberand sericin, which is the sticky material surrounding the fibroin. Silkfibroin is a β-keratin material known to be a reliable suture materialwith mid-range degradation kinetics and solid mechanical strength. Silkfibroin which has unique biocompatibility features including itsdegradation products. Silk fibroin degrades to amino acids, which arenatural to the body.

Chitosan is a naturally occurring polysaccharide composed of alternatingacetylated and deacetylated D-glucosamine residues. The polysaccharideis derived from the deacetylation of the exoskeleton of crustaceans andhaving the chemical formula Poly-(1-4)-2-Amino-2-deoxy-β-D-Glucan asreflected in the following structure:

Chitosan has been used clinically in hemostatic wound dressings and isemerging as a promising constituent of novel biocompatible matrices intissue engineering. (Khor E, Lim L Y. “Implantable applications ofchitin and chitosan.” Biomaterials 24(13) (2003) 2339-2349). Chitosandegrades to sugars, also part of the body's metabolism and easilyincorporated into other metabolic products. Since the degradation of theproduct does not change the local pH, there is no adverse effect such asobserved with polyglucolic or polylactic acids, whose degradationreduces the local pH and causes a sterile inflammatory response.

Silk fibroin combined with chitosan has been found to be useful inrepair of tissue defects. See e.g. Gobin A S, Butler C E, and Mathur AB. “Repair and regeneration of the abdominal wall musculofascial defectusing silk fibroin-chitosan blend.” Tissue Eng 12(12) (2006) 3383-3394.Silk fibroin can be blended with chitosan at different ratios including25:75, 50:50, 60:40, or 75:25, with the different ratios providingdifferent physical characteristics.

Example 6

Silk fibroin-chitosan scaffolds were prepared in a series of steps. Thesericin coating of raw silk fiber was removed via degumming. Solutionsof 0.25% (w/v) sodium dodecyl sulfate (Sigma-Aldrich, St. Louis, Mo.)and 0.25% (w/v) sodium carbonate (Sigma-Aldrich) were dissolved andheated to 100° C. Silk was added at 1:100 w/v, heated for 1 hour,followed by draining of the alkaline soap solution. Degummed silk wasrinsed in running distilled water, air-dried, and then dissolved incalcium nitrate tetrahydrate-methanol (molar ratio 1:4:2calcium:water:methyl alcohol) at 65° C. The silk fibroin (SF) wasdissolved at 10% (w/v) concentration over a 3-h period with continuousstirring.

Chitosan (CS) solution was prepared by 2% acetic acid dissolution ofhigh-molecular-weight chitosan (82.7% deacetylation; Sigma-Aldrich).Under continuous stirring, SF and CS solutions were combined forpreparation of 75:25 (v/v) SF:CS blend, followed by mixing for 30minutes, and then dialysis (molecular weight cutoff, 6-8 kDa) for 4 daysagainst deionized water.

Forty ml of SFCS blend solution was added to a glass Petri dish and thennon-directionally frozen overnight at −80° C., followed by 2-daylyophilization. Dry samples were treated in a 50:50 (v/v) methanol:1Nsodium hydroxide (NaOH) solution for 15 minutes for SF crystallizationand CS neutralization. Methanol:NaOH was then replaced by 1 N NaOH for12-18 hours. NaOH was removed by dilution in phosphate-buffered saline(PBS, 1×) with sequential changes of solution hourly for 4 hours andthen quarter-hourly until pH equilibration at 7.0. Samples weresterilized with 70% ethanol immersion for 12-18 hours, and subsequentlyrinsed in sterile PBS prior to in vitro cell seeding and subsequent invivo engraftment. Final scaffold thickness was 1.5 mm.

In other embodiments, thin sections of SFCS were fabricated using a75:25 SFCS blend. One ml of a 75:25 SFCS blend was added to a flexipermmold adhered to a glass slide and oven dried at ˜60° C. overnight. TheSFCS film was treated with 50:50 (v/v) methanol:1N NaOH for 15 minutesfor SF crystallization and CS neutralization. The methanol:NaOH wasreplaced by 1 N NaOH for 12-18 hrs and the NaOH removed by dilution inPBS 2-3× for 30 minutes until the pH equilibrated at 7.0. Again the SFCSfilm was sterilized by 70% ethanol immersion for 12-18 hours. Afterrinsing with sterile PBS the SFCS scaffold was ready for seeding.

In an experiment to test the functionality of pre-seeded silk-fibroingrafts made as described above versus unseeded grafts, human ADSC-seededSFCS were tested as a cyto-prosthetic hybrid for reconstructive supportin a murine cutaneous wound healing model. ADSC-SFCS were found tosupport the delivery and engraftment of stem cells as well asdifferentiation into fibrovascular and epithelial components. Humanadipose tissue was obtained from elective body contouring procedures andthe tissue digested with a solution of 0.07% Blendzyme 3 (F. Hoffman-LaRoche Ltd, Basel, Switzerland) with mild agitation at 37° C. for 60minutes, passed through a 40 μm filter and finally selected based onadherence to T75 tissue culture flasks at 24 hours. Cells were grown inalpha MEM medium supplemented with 20% fetal bovine serum, 2 mML-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells wereincubated in a 5% CO₂-containing chamber at 37° C. with medium changedevery 3 days. ADSCs between passages 1 and 8 were used for allexperiments. ADSCs used in these experiments have been previouslycharacterized and the multi-lineage differentiation potential of thesecells demonstrated. (Bai X, et al. “Electrophysiological Properties ofHuman Adipose Tissue-Derived Stem Cells.” Am J Physiol Cell Physiol293(5) (2007) C1539-50).

For seeding of the grafts, six-mm diameter SFCS grafts were placedcompletely covering the well bottom in 96-well plates and the graftswere covered with 200 μl aliquots of medium alone in the SFCS group andwith equal volume of cell suspension containing 1×10⁵ ADSCs/cm² in theADSC-SFCS group. Grafts were incubated under standard culture conditionsfor 24 hours after which overlying medium or cell suspension wasaspirated. The grafts were flipped to place the opposite surface facingup, and the corresponding medium or cell suspension solution was placedon the other side. Grafts were then incubated for 24 hours andtransferred to the operating suite for surgical engraftment. Once on theoperative field, grafts were transferred to a sterile 6-well plate andwashed gently in 2×500 μl aliquots of PBS to remove any non-adherentcells or medium. For the main study ten animals were randomized to oneof three treatment groups: no graft, SFCS alone or ADSC-SFCS. Animals ineach group received one 6 mm punch lesion and a graft-based repairdepending on group randomization.

Wound closure was measured by planimetric analysis and revealed a woundclosure at post-op day 6 of 46±15% in the control group receiving nograft, 58±9% in the SFCS group and 72±5% in the ADSC-SFCS group(p≦0.05). Post-op day 8 values were 55±17% in the no graft group, 75±11%in the SFCS group and 90±3% in the ADSC-SFCS group (p≦0.05). Wound bedanalysis of fresh tissue mounts demonstrated a markedly enhanced extentof wound closure in the ADSC-SFCS group in comparison to both the SFCSand no-graft control groups at post-op day 9. Close inspection of imagesunder intense illumination revealed an apparent more robust invasion ofvascular tissue, characterized by hyperemia, in the SFCS and ADSC-SFCSgroups versus no-graft controls. Furthermore, the extent of vascularinfiltration of the surrounding tissue in the region of the operativesite was greater in qualitative magnitude in the ADSC-SFCS group versusthe SFCS group (data not shown). Mean micro-vessel density in theADSC-SFCS group at 2 weeks post-op was 7.5±1.1 vessels/high power field,while density in the SFCS group at two weeks was 5.1±1.0 vessels/highpower field (p≦0.05). There was no evident inflammatory infiltrate (nopolymorphonuclear cell infiltration, no giant cells noted) on any H&Estains of wound bed biopsies at two weeks, indicating excellentbiocompatibility of engrafted SFCS. FIGS. 13 and 14 are scanningelectron micrographs of SFCS seeded with ADSC. As can be seen in theimages, the ADSC preferentially adhere to regions having surfacestructure or micro-roughness.

This study showed that a 75:25 silk fibroin-chitosan blend acts as ascaffold for the seeding and in vivo delivery of human adipose-derivedstem cells and confers the physiologic benefits of accelerated woundclosure. Histological analysis showed that the ADSCs engraft,proliferate and differentiate into fibroblastic, vascular, andepithelial phenotypes in their new microenvironment and that such seededgrafts potentiate local vascular ingrowth. The culture-expanded ADSCswere shown to adhere to a SFCS substrate in the range of 75% adhesion byone hour post-seeding, with adherent stem cells occupying both surfaceand three-dimensional elements of the scaffold. The clear engraftment ofADSCs into regenerating tissue in this study differs from previousreports where the engraftment of therapeutically introduced mesenchymalstem cells has been either un-observable or observable only at lowlevels. (Prockop D J. “‘Stemness’ does not explain the repair of manytissues by mesenchymal stem/multipotent stromal cells (MSCs)” ClinPharmacol Ther. 82(3) (2007) 241-243).

Successful seeding of grafts has been conducted with fresh SVF cells aswell and provides the further benefits of larger numbers of cells loadedon the graft and a greater population diversity as well.

Seeding of the Matrices

Cells for regenerative medicine can be delivered as a suspension,including delivery of suspensions of cells to specific targetcompartments. For example, one of the present inventors has disclosed aprocess for repairing tissue by delivering stem cells to a site of thetissue to be repaired through the vascular tree or the pre-existingdistribution trees in the body and that such focal application of cellsis beneficial, for example for repair of a patient's heart, brain,liver, kidney, pancreas, lungs, nerves, and muscles. (Alt, E., U.S. Pat.No. 6,805,860 and related CIP application issuing as U.S. Pat. No.7,452,532).

While delivery of cell suspensions may be indicated in certain tissuerepair or regeneration applications, in other indications it may bedesirable to localize the cells in high concentration on a matrix orscaffold in order to provide a locally enriched population of desiredcells and to retain their presence at the site of desired action untilhealing is well underway. Provided herein are methods and compositionsthat are able to seed and retain cells of interest on a biocompatiblescaffold. Also provided are methods to most effectively use the scaffoldin conjunction with a freshly isolated cellular preparation that avoidsa need for culturing of the cells. In other embodiments, methods andcompositions are provided to selectively enrich for cells that aredesired in specific tissue regeneration applications based on theiradherence to different biomaterials, while removing cells that may bedetrimental to the tissue repair or regeneration process.

In one embodiment of the invention, the reparative cell population isisolated as above and immediately seeded onto a biocompatible matrix. Inother embodiments, the stromal vascular fraction is isolated and subjectto culture to isolate the adherent cells that have been characterized asmesenchymal stromal cells. The adherent cells are seeded onto scaffoldsand, as further shown herein, will adhere to the scaffold within 2 hoursand preferably within one hour.

In one embodiment of the invention, the reparative cell isolationapparatus such as that depicted in FIG. 6 includes an in-line seedingchamber 180. For example, as described in FIG. 7, the SVF fraction isremoved from under the lipid containing layer in the lipid separatingchamber 140 and the SVF cells are pulled or pushed into the seedingchamber 180 by the action of a pump (not shown) that can be place up ordownstream of the seeding chamber. For example, where a porous scaffoldis used, the cells can be forced into rapid contact with the surface ofthe scaffold when the fluid medium containing the cells is pulledthrough the scaffold. After a contact time whereby a desired % of thedesired cells have adhered to the scaffold, nonadherent cells and debrisare removed through nonadherent conduit 187. In other embodiments, theseeding chamber 180 is a separate apparatus or unit from the isolationapparatus 100.

Optionally, a porous cell retentive membrane having pore size between0.2 and 5 μm, preferably between 1 and 3 μm is situated immediatelybeneath the porous cell scaffold. The cells are forced into rapidcontact with the surface of the scaffold when the fluid mediumcontaining the cells is pulled through the scaffold and retained incontact or close proximity to the porous cell scaffold by the porouscell retentive membrane. Media to enhance cell adherence of desiredreparative cells to the membrane may be introduced at this time. Mediamay include, but not be limited to, cell culture media supplemented withthe patient's own plasma or serum, carbohydrates such as carboxymethyldextran or iron dextran, or cold insoluble globulin. After a contacttime of 5 minutes to 2 hours, non-retained cells and debris are removedthrough conduit 187. Media or porous membrane composition that mayfurther support cell survival following implantation in situ may also beincorporated for example inclusion of local oxygen delivery component(U.S. Pat. No. 7,160,553). In other embodiments, the seeding chamber 180is a separate apparatus or unit from the isolation apparatus 100.

In one embodiment, the seeding chamber 180 is a disposable unit that isloaded with the scaffold 185 before the cell isolation begins. The shapeand size of this scaffold is adaptable to its intended clinical use,including shape and dimension and two or three dimensionalconfiguration.

In one embodiment, as depicted in FIG. 7, procedural steps in use of aseeding chamber include the following: SVF or MSC are conveyed to aseeding chamber which is adapted for use depending on a configurationand cell selection criteria tailored to the tissue to be repaired. Thecells are introduced into the seeding chamber in a way that promotesphysical interaction between the cells and the matrix material of thescaffold and a certain contact time between 15 and 120 minutes isallowed for desired cells to attach and/or migrate into structure of thescaffold, following which cells that have not adhered to the scaffoldare evacuated or washed from the seeding chamber.

In one embodiment of the invention as depicted in FIG. 8, a series ofselective seeding chambers are utilized in serial fashion. For example,SVF cells are isolated and directed into selective seeding chamber Awherein scaffold A is adapted to selectively bind a population of cellson the basis of an “A” ligand. Non-adherent cells lacking the “A” ligandare washed from the A chamber and directed to selective seeding chamberB, which contains selective scaffold B, which is adapted to selectivelybind a population of cells on the basis of a “B” ligand. Non-adherentcells lacking both the “A” and “B” ligand are washed from chamber B andcollected. Adherent cells in chambers A and/or B may be collected afterrelease from the scaffolds. The selective seeding chambers may be usedfor either positive or negative selection in the generation of specificcell populations.

In one embodiment of the invention, introduction of the reparative cellsonto the scaffold is facilitated by pulling the cells through a porousscaffold in such a way that physical contact between the cells and thesurface of the scaffold is promoted. For example, the seeding can beenhanced by vacuum or other physical force applied to the cells to forcethe cells into physical contact with the matrix or scaffold. In oneembodiment of a seeding chamber and method of use thereof, the cells areintroduced to the chamber prior to an attachment period of less than 2hours. For use, the scaffold is removed from the seeding chamber andapplied to or implanted at the target site on a human or animal patient.

FIG. 9A depicts one embodiment of a seeding chamber 180 wherein cells tobe seeded onto scaffold 185 are introduced into seeding chamber 180through inlet 181. In the depicted embodiment, scaffold 185 is mountedin the chamber in such a way that a fluid entering the chamber frominlet 181 may not leave through outlet 186 without passing through thescaffold 185. In the depicted embodiment, the scaffold 185 is supportedby porous support 190. The chamber is designed so that no fluids canpass to outlet 186 without going through the scaffold 185. In this way,a pump or other partial vacuum source (not shown) disposed in fluidcommunication with outlet 186 is able to pull fluids through thescaffold and any cells entering the chamber will be rapidly pulled intocontact with the scaffold. In the embodiment depicted in FIG. 9A, thetop of the chamber includes a plurality of ribs 194 that are arrayed toconvey fluid entering the chamber down channels 192 such that cells arerelatively evenly dispersed over the scaffold surface. The top andbottom aspects of the chamber are connected by a resealable closure suchas threaded closure 195, which enables ready opening of the chamber forinsertion of the scaffold as well as removal of the cell seededscaffold.

FIG. 9B depicts an alternative embodiment of a seeding chamber 180.Again cells to be seeded onto scaffold 185 are introduced into seedingchamber 180 through inlet 181. In the depicted embodiment, the upperportion of the chamber includes a port 187 whereby media can beexchanged, additive introduced, and cells that do not adhere to thescaffold can be drawn off after an incubation period. In the embodimentdepicted in FIG. 9B, cells are relatively evenly distributed over thescaffold by a plurality of channels 192 that are manufactured into thelid or top of the chamber. Also in the depicted embodiment, the scaffold185 is supported by a plurality of supports 196. Porous support 190 maynot be necessary or desired. In one embodiment, the supports 196represent the upper aspect of a grid or spiral or labyrinthine formhaving a plurality of drainage holes. The flow of fluid through thescaffold is essentially perpendicular to the plan of the scaffold asdepicted by the arrows.

FIG. 10 depicts a seeding chamber according to one embodiment of theinvention. Cells to be seeded onto porous scaffold 185 are introducedinto seeding chamber 180 through inlet 181. In the depicted embodiment,porous scaffold 185 is mounted in the chamber in such a way that a fluidentering the chamber from inlet 181 may not leave through outlet 186without passing through the scaffold 185. In one embodiment, a sealingring 182 insures that no fluids can pass to outlet 186 without goingthrough the scaffold 185. In this way, a pump or other partial vacuumsource (not shown) disposed in fluid communication with outlet 186 isable to pull fluids through the scaffold and any cells entering thechamber will be rapidly pulled into contact with the scaffold.

After a predetermined attachment period, wash outlet 187 is opened andnon-adherent cells and debris are pulled out of the chamber. If desired,a selective red blood cell lysis can be undertaken in the chamber, usingfor example hypotonic solutions, surfactants, ammonium chloride (155 mMNH₄Cl, 10 mM KHCO₃), carbamates (U.S. Pat. No. 7,300,797) etc., withoutcompromising the viability of the nucleated cells.

Typically, as depicted in FIG. 10 the chamber 180 is constructed suchthat it can be opened after cells have been deposited on the scaffoldand the scaffold removed for implantation or further processing.

Example 7

In one embodiment of the invention, the reparative cell population iscontacted with the scaffold for less than about 90 minutes while inother embodiments, a contact time of approximately 60 minutes or evenless is sufficient. A relatively short contact time was found to beeffective in inducing the adherence of greater than 90% of adiposederived stem cells to a bioabsorbable ADM matrix. ADSCs were isolatedfrom discarded adipose tissue obtained at body contouring proceduresaccording to standard methodology for isolating mesenchymal stromalcells. Briefly, lipoaspirate was washed with sterile phosphate-bufferedsaline (PBS). Washed aspirates were treated with a mixture ofcollagenase and neutral protease in PBS for 30 min at 37° C. withagitation. The enzyme was inactivated with an equal volume of DMEM/10%fetal bovine serum (FBS) and the cells were collected by centrifugationfor 10 min at low speed. The cellular pellet was resuspended in DMEM/10%FBS and filtered through a 100 μm mesh filter to remove debris. Thefiltrate was centrifuged as before and plated onto conventional tissueculture plates DMEM/20% FBS for culture. Non-adherent cells were removedafter 24 hours by aspiration and the adherent cells were expanded inculture with media changes at 3 day intervals. Cells at passage 1-8 wereused for experiments.

ADM of thickness 0.4 to 0.8 mm was obtained (AlloDerm®, LifeCell,Branchburg, N.J. and FlexHD™, MTF, Edison, N.J.). Passage 1-5 cellsuspensions in growth medium were seeded into multi-well plates coveredwith ADM with the papillary dermis side facing up at a density of5.0×10⁴ cells/cm² for histology, 7.5×10⁴ cells/cm² for quantitativestudies, and 1.0×10⁶ cells/cm² for scanning electron microscopy (SEM).

Adherence was quantified by fluorescent cell counts at 15, 30, 60 and120 minutes. Specimens for histology and SEM were seeded and incubatedunder standard culture conditions for 24 hours. Specimens werehematoxylin and eosin (H&E) stained. SEM was performed using adaptationsof established methodology.

The ADSCs were negative for the leukocyte markers CD45 and CD11b, andpositive for the intermediate filament nestin, CD44, CD90, and CD105.Quantitative adhesion experiments revealed very rapid adherence of ASCto the ADM. It was found that 543±62 cells/mm² adhered at 15 minutes(71%), 688±69 cells/mm² adhered at 30 minutes (92%), 713±63 cells/mm²adhered at 60 minutes (94%), and 727±54 cells/mm² adhered at 120 minutes(96%). H&E and SEM analysis confirmed stem cell adhesion to ADM.

This experiment showed that the AdSC components of the reparative cellmixture are able to rapidly bind to a bioabsorbable ADM matrix. Thus,the ADSC present in the reparative cell mixture can be seeded onto amatrix and be available for implantation in one operative procedure. Theobservation that ADSCs adhere to ADM at high rates within a two-hourtime frame is significant. Furthermore, SEM identified active attachmentof stem cells to ADM with the extension of microvilli and lamellopodiaacting as focal anchorage points.

Example 8

The rapid seeding of SFCS scaffolds was also tested to confirm thefeasibility of point of care seeding of scaffolds. ADSC were isolated asdescribed in Example 5 and seeded onto SFCS scaffolds produced asdescribed in Example 4. For adhesion studies, cells were transfectedwith green fluorescent protein as described by Zhang et al.“Transduction of bone-marrow derived mesenchymal stem cells by usinglentivirus vectors pseudotypes with modified RD114 envelopeglycoprotains” J. Virol. 78 (2004) 1219-29. Adherence was qualified bydirect triplicate counts of adherent GFP positive cells.

It was shown that 75% of a population of ADSC would bind to a SFCSscaffold within 60 minutes. Specifically, in one experiment the numbersof adherent ADSCs to SFCS with time were as follows:

369±53.16 adherent cells/field (49.20±7.09% of seeded cells) at 15minutes

365±81.30 adherent cells/field (48.78±10.84% of seeded cells) at 30minutes

566±75.05 adherent cells/field (75.46±10.01% of seeded cells) at 60minutes

572±131.33 adherent cells/field (76.30±17.51% of seeded cells) at 120minutes

SEM findings provided qualitative confirmation of the above adhesiondata that was based on GFP fluorescent counts. A scatter distribution ofadherent stem cells was observed at 15 minutes post-seeding, withadherent cells focused on the parallel sheet edge and around fiberprojections of the surface. Cell spreading was noted by 30 minutes andby 60 minutes cells were beginning to adhere to the flat and smoothregions of the SFCS sheet that were initially minimally populated. By120 minutes a full blanketing was observed with progressive spreading ofcells on flat regions and the dense occupation of fiber convolutions andmicro-enclaves at the junctions of fibers and sheets. The resultsobtained with ADC isolated with a step including plastic adherence wereconfirmed with freshly isolated cells. FIG. 17 presents a SEM image offreshly isolated SVF cells adhering to SFCS after 15 minutes ofincubation. As can be seen, many of the cells have elaborated microvilliand lamellopodia by such time.

The results showed a preference of the cells for adherence to roughtopography and pointed to this preferential structural character ofparticularly desirable scaffolds. Thus, in one embodiment of theinvention a method of inducing adherence of reparative cell populationsto tissue scaffolds includes generation of a rough surface topography tothe scaffold wherein the surface is characterized by surfaceirregularities occurring on a scale of one to one hundred μm.

Preorientation of ADC and SVF:

In one embodiment of the invention, adherent cells from humanlipoaspirate isolated in accordance with Example 1 and 2 are exposed toselect induction media to preorient responsive cells into a desireddifferentiation track prior to administration into the patient. Thefollowing are non-limiting examples of induction media that are known todrive the differentiation of cells into particular lineages by prolongedculture in the media.

Examples of Media for Cell Pre-Orientation

Lineage Component Conc. Adipogenic DMEM, low glucose Fetal bovine serum(FBS) 10% L-glutamine 2 mM Penicillin/Streptomycin L-Ascorbic acid 100μM 1-methyl-3-isobutylxanthine, 0.5 mM (IBMX) Dexamethasone 1 μMIndomethacin 100 μM Insulin human recombinant 10 μg/ml Assess subsequentadipogenesis by Oil Red O staining

Lineage Component Conc. Chondrogenic DMEM, high glucose FBS 10%Dexamethasone 0.1 μM Ascorbate-2-phosphate 25 ug/ml Insulin, bovine 10μg/ml TGFβ-3 (R&D) 10 μg/ml Sodium pyruvate 1 mM Non-essential aminoacids Proline 0.M Transferrin 5.5 μg/ml Sodium selenite 5 ng/ml LinoleicAcid 4.7 ng/ml Bovine Serum Albumen 0.5 mg/ml (BSA) Assess chondrogeneisby expression of proteoglycan or collagen II using histochemistry orimmunohistochemistry staining.

Lineage Component Conc. Endothelial DMEM, (low glucose) FBS 2%Penicillin 10 U/ml Streptomycin 100 ug/ml VEGF 50 ng/ml L-glutamine 2 mMAssess endothelial like cells by detection of vWF byimmunohistochemistry.

Lineage Component Conc. Hepatogeneic DMEM, (1 g/L glucose) FBS 1% bFGF(Chemicon) 10 ng/ml aFGF (Chemicon) 20 ng/ml EGF 10 ng/ml HGF (R&D) 20ng/ml Insulin-transferrin-selenious acid 1% (ITS-BD Biosciences)Oncostatin M (OSM) 10 ng/ml Assess hepatogenesis by detection of albuminby immunofluorescence.

Lineage Component Conc. Myogenic DMEM, (low glucose) FBS 10% Horse Serum(HS) 5% Penicillin/streptomycin 1% Hydrocortisone 50 μM Assessmyogenesis by detection of myosin by immunofluorescence.

Lineage Component Conc. Neurogenic DMEM, F12 FBS 1% B27 (Invitrogen) 2%L-ascorbic acid 50 μM Insulin 5 μg/ml bFGF (Chemicon) 10 ng/ml bEGF 10ng/ml NGF (R&D) 10 ng/ml 2-mercaptoethanol 1 mM forskolin 10 μM cAMP 2mM 1-methyl-3-isobutylxanthine, 0.5 mM (IBMX) indomethacin 200 μM Assessneurogenesis by detection of microtubule-associated protein-2 (MAP-2) byimmunofluorescence.

Lineage Component Conc. Osteogenic FBS 10% Dexamethasone 0.1 μML-Ascorbic acid 0.2 mM β-glycerol phosphate 10 mM Assess subsequentmineralization by calcium deposit by staining with Alizarin Rd S

Applications

The cellular preparations of the present invention including thedifferent biocapatable matricies can be applied to subjects for variouscell therapeutic purposes. Such cell therapies generally refer to theregeneration and/or repair of injured or diseased tissue. Non-limitingexamples include wound healing (infected and non-infected), bonefracture healing, treatment of non-healing ulcers, hernia repair, tendonrepair, plastic surgical indications including skin grafting, cartilageregeneration, including cartilage of the nose and pinna of the ear,engraftment after chemotherapy, rescue of retinal degeneration,treatment of ischemic disease (e.g., ischemic heart disease andperipheral arterial disease), treatment of nerve injury, filling ofheart aneurysms and of the atrial appendage, creation of an artificialbladder and bladder wall repair, repair and reconstruction ofintestines, and repair and reconstruction of vessels and associatedstructures.

Cardiac Applications: In one embodiment, reparative populations areseeded onto biomaterial matrices for the treatment of various physicaldefects of the heart including the roughly 1% of the population with anatrial septal defect which enables a shunt between the right and leftatrium. Other deficiencies are ventricular septum defect and patentforamen ovale (PFO) that are amenable by occlusion either by directsurgical techniques in suturing a cell seeded patch or by non-invasivecorrection in placing a cell seeded occluder. While all currentoccluders and materials to close such a patch are considered to be ofnon-absorbable materials such as Teflon®, Dacron®, stainless steel,Elgiloy® or Nitinol™, the present invention provides biomaterials whichare absorbable and coated with stem cells which allows a natural healingas certain of the included multipotent cells differentiate intofibroblasts as well as cardiomyocytes.

Another application for the biomaterial is to occlude the left auricleof the left atrium from which a good deal of thrombotic events can occurand previous experience has shown that the occlusion of such an auriclecan reduce the formation of thrombus and prevent the embolic events. Inanother embodiment, reparative cell coated biomaterial is used forrepair of aneurysms of the vascular structures, including aneurysms ofthe aorta and arteries. Placement can optionally be made from inside thevessel in the form of a covered stent, which would be a combination of ascaffold and a coated biomaterial membrane.

All publications, patents and patent applications cited herein arehereby incorporated by reference as if set forth in their entiretyherein. While this invention has been described with reference toillustrative embodiments, this description is not intended to beconstrued in a limiting sense. Various modifications and combinations ofillustrative embodiments, as well as other embodiments of the invention,will be apparent to persons skilled in the art upon reference to thedescription. It is therefore intended that the appended claims encompasssuch modifications and enhancements.

We claim:
 1. A cell-seeded tissue graft comprising a reparative cellpreparation seeded onto a porous scaffold, wherein the tissue graft isprepared by: isolating a fresh stromal vascular fraction (SVF) from anadipose tissue of a patient by a process including enzymaticallydigesting the adipose tissue; applying the fresh SVF cells to the porousscaffold without subjecting the fresh SVF cells to plastic adherence;and rinsing the porous scaffold to eliminate cells that are unbound bythe porous scaffold, thereby generating a tissue graft comprising areparative cell preparation in less than about 4 hours from removal ofthe adipose tissue from the patient.
 2. The cell-seeded tissue graft ofclaim 1, wherein the porous scaffold is characterized by a micro-roughcell attachment surface that has surface irregularities at a periodicityof 1-20 μm.
 3. The cell-seeded tissue graft of claim 1, wherein thecell-seeded tissue graft is generated at a point-of-care and isimplanted into the patient without culturing the cell-seeded tissuegraft.
 4. The cell-seeded tissue graft of claim 1, wherein thecell-seeded tissue graft is cultured to expand populations of cellsseeded on the graft prior to implanting into the patient.
 5. Thecell-seeded tissue graft of claim 1, wherein the SVF cells are pushedinto contact with the porous scaffold by pressure.
 6. The cell-seededtissue graft of claim 1, wherein the SVF cells are pushed into contactwith the porous scaffold by a partial vacuum.
 7. The cell-seeded tissuegraft of claim 1, wherein the SVF cells are incubated with an inductivemedia before, during or after being applied to the porous scaffold. 8.The cell-seeded tissue graft of claim 7, wherein the inductive media isadapted for generation of one or more of adipocytes, chondrocytes,endothelial cells, hepatocytes, myoblasts, neurons, and osteoblasts. 9.The cell-seeded tissue graft of claim 1, wherein the porous scaffold iscomprised of a biocompatible or a biodegradable material.
 10. Thecell-seeded tissue graft of claim 9, wherein the biocompatible materialis selected from the group consisting of: polytetrafluoroethylene, wovenpolyester, spun silicone, open foam silicone encased in microporousexpanded PTFE, stainless steel, polypropylene, polyurethane,polycarbonate, nickel titanium shape memory alloys andcobalt-chromium-nickel alloys, and combinations thereof.
 11. Thecell-seeded tissue graft of claim 10, wherein the biodegradable materialis selected from the group consisting of: silk fibroin-chitosan,acellular dermal matrices, collagen, polyglactin, hyaluronic acid, andresorbable silica gel matrix.
 12. The cell-seeded tissue graft of claim2, wherein the surface irregularities of the porous scaffold are createdby treatment of at least one cell attachment surface of the scaffold byone or more of embossing, blasting, plasma etching, by controllingpolymerization or drying processes, by heat application, by chemicaletching, and by coating or printing.
 13. The cell-seeded tissue graft ofclaim 2, wherein at least one surface of the porous scaffold ischaracterized by a spongy texture formed by subjecting the nascentscaffold material to a partial vacuum during polymerization or drying.14. The cell-seeded tissue graft of claim 1, wherein the cell-seededtissue graft is adapted and configured to treat one or more of: woundhealing, burns, bone fractures, cosmetic defects, cartilage damage,tendon damage, ulcers, fistulas, hernias, retinal degeneration,treatment of ischemic disease, nerve injury, aneurysms, bladder wallrepair, intestinal injury, and vascular vessel repair.
 15. Thecell-seeded tissue graft of claim 1, wherein an adherence agent selectedto promote adherence of desired cell types is applied to the porousscaffold prior to cell-seeding.
 16. The cell-seeded tissue graft ofclaim 15, wherein the adherence agent is selected from the groupconsisting of: autologous plasma or serum and components thereof, coldinsoluble globulin, carboxymethyl dextran, iron dextran, and hyaluronicacid and polymers thereof.
 17. A cell-seeded tissue graft comprising areparative cell preparation seeded onto a porous scaffold, wherein thetissue graft is prepared by: isolating a fresh stromal vascular fraction(SVF) from extraction fluid of a lipoaspirate from a patient; applyingthe fresh SVF cells to the porous scaffold without subjecting the freshSVF cells to plastic adherence; and rinsing the porous scaffold toeliminate cells that are unbound by the porous scaffold, therebygenerating a tissue graft comprising a reparative cell preparation inless than about 4 hours from removal of the adipose tissue from thepatient.